Cytomegalovirus (CMV) is the leading viral cause of congenital birth defects in 1%-3% of live births in the United States. Half the mothers developing first-time infection during pregnancy will transmit virus to the fetus; 25% of newborns will have congenital disease and permanent birth defects.
Maternal low-avidity antibodies to CMV are a key indicator of possible fetal infection. ELISA assays to identify women at risk for primary maternal CMV infection are marketed by Radim (Italy) and BioMerieux (France) and other companies. These assays quantify maternal CMV IgG avidity for diagnosis of primary infection acquired during or shortly before gestation. Low-avidity antibodies indicate primary infection with 50% chance of fetal infection. However, women with moderate IgG avidity are not necessarily protected against congenital infection in the fetus, although damage is milder.
Transforming growth factor-β1 (TGF-β1), a multifunctional cytokine, plays a central role in cell proliferation, migration, and synthesis of extracellular matrix (ECM) in the endothelium (Lebrin et al., Cardiovasc Res, 65:599-608 (2005)). In most cell types, TGF-β1 signals through the type I receptor activin receptor-like kinase 5 (ALK5). In addition to expressing ALK5, endothelial cells express a second TGF-β1 receptor, the type I receptor ALK1. When activated, ALK1 induces phosphorylation of the nuclear effectors Smad 1 and Smad5, which promote endothelial cell proliferation and migration (Chen, Y. G. and Massague, J., J Bioi Chem, 274:3672-3677 (1999)). In contrast, activated ALK5 induces Smad2 and Smad3 phosphorylation, leading to the inhibition of endothelial cell proliferation. TGF-β1 is secreted as an inactive, noncovalent complex with latency-associated peptide and requires activation before it can bind to its receptors. Reported mechanisms of TGF-β1 activation include cleavage by metalloproteinases or plasmin and binding to thrombospondin 1 or either of the integrins αvβ6 and αvβ8 (Lebrin et al., Cardiovasc Res, 65:599-608 (2005); Rifkin, D. B., J Biol Chem, 280:7409-7412 (2005); Munger et al., Cell, 96:319-328 (1999); Mu et al., J Cell Biol, 157:493-507 (2002); Crawford et al., Cell, 93:1159-11170 (1998); Annes et al., J Cell Sci, 116:217-224 (2003)). One of the in vivo activators of TGF-β1 is integrin αvβ6 (Rifkin, D. B., J Biol Chem, 280:7409-7412 (2005); Munger et al., Cell, 96:319-328 (1999)). This activation model is particularly interesting because integrin αvβ6 is expressed principally on epithelial cells, which are very sensitive to TGF-β1-mediated growth inhibition. Integrin αvβ6 is strongly up-regulated at sites of epithelial repair and inflammation in lung and kidney (Breuss et al., J Cell Sci, 108:2241-2251 (1995)), and also because of the overlap of the phenotypes of TGF-β1 and integrin β6 subunit-deficient mice. Mice lacking the β6 subunit show increased inflammation and decreased fibrosis, both of which processes are strongly regulated by TGF-β1 (Munger et al., Cell, 96:319-328 (1999); Huang et al., J Cell Biol, 133:921-928 (1996); Hahm et al., Am J Pathol, 170:110-125 (2007)).
Recent work has provided evidence for the induction of TGF-β1 in a variety of cells and tissues on CMV infection. TGF-β1 was released in increasing amounts from splenocytes infected with rat CMV in vitro (Haagmans et al., J Gen Virol, 78:205-213 (1997)). TGF-β1 protein was increased in alveoli and stromal cells in rat lungs, spleen, and liver after radiation-induced immune suppression of CMV-infected rats (Haagmans et al., J Gen Virol, 78:205-213 (1997)). Furthermore, CMV infected murine astrocytes increased TGF-β1 transcription and protein levels (Kossmann et al., J Infect Dis, 187:534-541 (2003)). In human kidney allografts, CMV proteins and DNA were associated with locally increased TGF-β1 in tubuli and arterial endothelium long after viral clearance from the blood (Helantera et al., Transplantation, 79:379 (2005)). Brain biopsy specimens from AIDS patients with CMV encephalitis were found to contain viral inclusions that co-localized with TGF-β1 protein in cells with astrocyte-specific glial filaments (Kossmann et al., J Infect Dis, 187:534-541 (2003)). In addition, TGF-β1 induction in human fibroblasts has been shown to involve the transactivation of its promoter by immediate-early 2 protein through an Egr-1 consensus site by binding the zinc finger domain of Egr-1 (Michelson et al., J Virol, 68:5730-5737 (1994); Yoo et al., J Viral, 70:7062-7070 (1996)). Although the evidence suggests that TGF-β1 may be directly involved in CMV pathogenesis, little is known about the cellular proteins involved in virus-mediated TGF-β1 activation, or what specific functional role it plays in vivo. In recent experiments, it was found that a subpopulation of freshly isolated human cytotrophoblasts from term placentas expressed integrin αvβ6, which activates TGF-β1 in vitro (Tabata et al., Placenta, 28:527-537 (2007)).
Currently, there are no commercial assays to detect fetal infection early in gestation or to predict symptomatic disease. In women with primary CMV infection in first trimester, ultrasound at midgestation may identify fetuses with intrauterine growth restriction (IUGR) and other disease anomalies. But these may not be apparent unless severe (e.g. microcephaly and calcification in the brain). Detection of viral DNA by PCR following amniocentesis at 20-22 weeks gestation indicates fetal infection; very high levels may be associated with symptomatic fetal disease (Pereira et al., J. Virol. 77:13301-13314 (2003)). At birth, congenitally infected babies secrete infectious virus in urine, viral DNA can be quantified, and infectivity evaluated in plaque assays. Blood from infants with symptomatic disease contains many genome copies of CMV DNA (>10,000/ml).
Until recently, there was no therapy to prevent symptomatic congenital disease. Nigro et al. reported that CMV hyperimmune globulin (HIG) (Biotest, Germany) was an effective treatment that reduced congenital disease from 50% to 3% in infants of women with primary infection treated with intravenous HIG (N Engl J Med 353:1350-62 (2005)). Echodensities, anomalies associated with placental insufficiently and IUGR can resolve following HIG treatment. Moreover, when administered soon after maternal seroconversion, fetal infection is prevented. Clinical trials for congenital. CMV infection are ongoing and proposed. Thus, diagnostic tests for early detection of fetal infection and to indicate treatment efficacy are desperately needed. Quantitative assays for biomarkers of viral replication in amniotic fluid could identify candidates for treatment and reduced levels could be objective indicators of efficacy.
Accordingly, the invention provides biomarkers of CMV replication that are detectable in amniotic fluid and that permit early detection of congenital infection before symptomatic disease.